Structural basis of bacterial effector protein azurin targeting tumor suppressor p53 and inhibiting its ubiquitination

Tumor suppressor p53 prevents tumorigenesis by promoting cell cycle arrest and apoptosis through transcriptional regulation. Dysfunction of p53 occurs frequently in human cancers. Thus, p53 becomes one of the most promising targets for anticancer treatment. A bacterial effector protein azurin triggers tumor suppression by stabilizing p53 and elevating its basal level. However, the structural and mechanistic basis of azurin-mediated tumor suppression remains elusive. Here we report the atomic details of azurin-mediated p53 stabilization by combining X-ray crystallography with nuclear magnetic resonance. Structural and mutagenic analysis reveals that the p28 region of azurin, which corresponds to a therapeutic peptide, significantly contributes to p53 binding. This binding stabilizes p53 by disrupting COP1-mediated p53 ubiquitination and degradation. Using the structure-based design, we obtain several affinity-enhancing mutants that enable amplifying the effect of azurin-induced apoptosis. Our findings highlight how the structure of the azurin-p53 complex can be leveraged to design azurin derivatives for cancer therapy.

5. Line 149-152, the authors stated that the SV-AUC experiment showed the fusion protein is in an equilibrium of monomer and dimer in solution. The claim is dubious, as the monomeric 39.2 kDa species is predominant. Could the authors elaborate more? Also, the main text mentions the SV-AUC experiments used the same protein concentration as crystallization, which is consistent with the 7.9 mg/mL in Supplementary Figure 3b legend. However, in the Methods section, line 429, the concentration is 0.75 mg/mL. Please clarify.
6. Line 159, is the 913 Å2 buried area on one molecule or the sum of buried areas on both azurin and p53? 7. In Figure 2a Leu 188 points away from the azurin molecule; yet in line 161 the authors wrote Leu188 interacts with a hydrophobic pocket of azurin. Could the authors elaborate on the role of Leu188? 8. Figure 3c: at the higher azurin concentration, the intensity of the p53 bands in both 2h and 4h incubation are less than those with no azurin or lower azurin concentration. This weakens the argument that with higher azurin concentration reduces p53 ubiquitination. Could the authors elaborate on this? In addition, did the authors observe a p53-Ub band in the anti-p53 blot? 9. Supplementary Figure 4: please provide the UniProt identifiers of the 20 azurin sequences. 10. The authors hypothesize that azurin competes with Bcl-xL for p53 dimer binding. Could the authors compare the binding affinities of azurin and Bcl-xL to p53? 11. From the text my understanding is azurin is Strep-tagged. The method also mentioned azurin is purified with Strep-Tactin columns. However, the cloning section describes azurin as His-tagged. Please clarify. Also, is it pET-15d or pET-15b vector?
12. Cloning and purification of GST-DBD (Figure 2b) are not described. Actually in the Methods section the pull-down assay is said to be between Flag-tagged p53-DBD, please clarify.
13. Line 344 and 399. MSAC-Ub, please spell out MSAC and cite proper references or manufacture protocols for FITC labeling of MSAC-Ub.
14. Line 400-401, there is no mentioning of hCOP1 autoubiquitination in the main text, please clarify.
15. Line 413, the space group is stated as P1212 here. In Table S1 it is C 1 2 1. Please clarify.

Reviewer #3 (Remarks to the Author):
This paper studies the structural basis of the interaction between a bacterial protein azurin and p53, using both solution NMR and X-ray. Based on the high-resolution structure of the protein complex, affinity enhancing mutations in azurin were designed and shown to enhance binding, inhibit p53 ubiquitination and enhance apoptosis through p53 stabilization. Overall, this work is very well done and has important application for improved azurin derived peptide drugs for cancer treatment. I highly recommend its publications. I only have a few minor concerns: 1. P53 is a tetramer in cell. The author may want to discuss the implication of DBD dimer interaction with azurin in this context. 2. Fig. 5 legend: panel descriptions do not correspond to the figure contents. Please correct. 3. Line 274, "apoptosis senescence": there should be a comma between these two words. 4. Line 275, "p53 inactivation": I rather use "p53 mutations" because there are gain-of-function p53 mutations. 5. Line 35, recommend rewording "officinal p28 region", for example, to "p28 region, which corresponds to a therapeutic peptide". 6. Line 40, "derivates" should be changed to "derivative" 7. Line 321-322: The first sentence of the paragraph sounds a little awkward. Reword to "Currently, major efforts are devoted to develop drugs to restore p53 stability and activity".

The paper describes structural and functional studies detailing the molecular interactions of 3
the bacterial effector protein azurin with tumor suppressor p53. Modulation of p53 plays an 4 important role in cancer therapy so this work has relevance to the wider community. 5 The authors report NMR studies demonstrating the regions of binding on the respective 6 proteins. These studies indicate weak binding interactions. Key residues involved in the binding 7 interactions are identified. The NMR structural data was used to design fusion proteins for 8 crystallography leading to the solution of a 2.2 Å X-ray structure of azurin-p53 fusion protein. 9 The interface of azurin with p53 is described in detail. Understanding of the protein-protein 10 interface allowed the investigation of the role of individual surface amino acids allowed the 11 identification of azurin mutants with improved binding affinity, The authors provide data that 12 shows the azurin mutants have downstream cellular effects mediated by stabilised p53. 13 However, the author should address the following issues: 50 suggest the structure does not reach the resolution, and/or a large region of the structure does 55 not fit well with the electron densities. The author should re-process the diffraction data and 56 re-refine the structure.      Interestingly, the crystal structure shows one azurin molecule binds to two p53 DBDs, in a way 229 similar to how the anti-apoptotic protein Bcl-xL binds to p53. The azurin binding interface on 230 p53 partially overlaps with COP1 footprint on p53. Mutations on the interface disrupts azurin: 231 p53 interaction and restores COPI-mediated p53 ubiquitination in vitro. The authors also 232 mutated interfacial residues in the hope to enhance azurin:p53 interaction and decreases p53 233 ubiquitination. This set of mutants interact with p53 with mildly enhanced affinity, but 234 drastically decrease p53 ubiquitination. Furthermore, when added to cells, these stabilizing 235 mutants lead to more cell apoptosis, demonstrating their potential in anticancer treatment. However, the author should address the following issues: 242

The authors reported the crystal structure resolution as 2.2 Å. However, in
Major concerns: 243 1. The authors reported the crystal structure resolution as 2.2 Å. However, in Table S1, the R-244 merge, R-meas, R-pim, and CC1/2 values are unusual for a 2.2 Å structure. Furthermore, the 245 Rwork/Rfree values of 26.5%/33.5% are too high for a 2.2 Å structure. The abnormal statistics 246 suggest the structure does not reach the resolution, and/or a large region of the structure does 247 re-refine the structure. 249 250 Re: Thanks very much for this comment. We carefully reprocessed the diffraction data and re-251 refined the structure, updated the statistics table of the crystal structure, and deposited our new 252 structure to the PDB server. The Rmerge/Rmeas values are 13.2%/14.7%, and the Rwork/Rfree 253 values are 21.1% /26.5%. To further validate the structure, we highlighted the interface and 254 overall structure fitting into the density map in Supplementary Fig 7. It shows that the structure 255 fits very well with the electron densities. resolution, which makes the dimer peak ambiguous. We have revised this figure and made it 308 in high resolution. Actually, we repeated this experiment two times. The dimer peak was 309 presented in both replicants. We also try to use higher concentrations, but the UV absorbance 310 from our UV detector on SV-AUC always overflowed, and it didn't give us a rational result. 311 In addition, the dimerization of p53_DBD (in the absence of DNA) has been characterized as 312 a very weak interaction by multiple published works, which explains our SV-AUC data that 313 the monomeric fraction is predominant compared to the dimer fraction at a relatively low 314 protein concentration. 315 We double-checked our experiments carefully. The sample concentration for SV-AUC 316 experiment is 0.75 mg/mL as the method part. We did run an SV-AUC experiment with 7.9 317 mg/mL sample, the UV absorbance overflowed as the concentration is too high. We have 318 already corrected our manuscript. Re: Thanks for the comment. We double-checked our re-refined structure and confirmed that 330 the Leu 188 points to azurin. The figure. 2a was revised. Actually, Leu 188 is very near to 331 azurin molecular, the distance between Leu 188 methyl groups to azurin is less than 4 Å, which 332 validates the hydrophobic interaction between these residues. 333 334 8. Figure 3c: at the higher azurin concentration, the intensity of the p53 bands in both 2h and 335 4h incubation are less than those with no azurin or lower azurin concentration. This weakens 336 the argument that with higher azurin concentration reduces p53 ubiquitination. Could the 337 authors elaborate on this? In addition, did the authors observe a p53-Ub band in the anti-p53 338 blot? 339 340 Re: Thanks for this comment. We did notice that the intensity of the p53 band is a little bit less 341 compared to the mixture without azurin due to sample loading. As p53 ubiquitination level is 342 dramatically different, we think that it is acceptable to back up our result. Also, we initially did 343 use anti-p53 blot to perform the ubiquitination assay. As the p53-Ub was much less than p53 445 446 447 in highly dynamic and flexible properties, the linker part and about 6 residues (4 residues 51 located at the C-terminus of DBD and 2 residues at the N-terminus of azurin) connected to the 52 linker part are invisible. This feature makes us disable tracing the exact linker position. 53 The distance between the visible C-terminus of DBD_A and the visible N-terminus of azurin_A 54 is 58 Å. The GS-linker residues plus the invisible and flexible terminal residues are about 26 55 residues. Theoretically, the average length of a residue backbone in a disordered peptide is 56 about 3.6 Å, so we think the linker length is sufficient to cover the distance. Although the C-57 terminus of DBD_A (top AU) is closer to the N-terminus of azurin_A in the bottom AU while 58 there is no direct interaction between these two molecules, so we don't think these two 59 molecules belong to the same fusion construct.